This invention relates to synthetic peptides having hypocalcemic activity similar to that of natural caicitonins of various species. It relates specifically to synthetic calcitonin peptides having a hypocalcemic potency when administered to humans greater than that of human calcitonin and/or having less immunogenicity in humans than calcitonins from foreign species. It relates as well to synthetic procedures for producing these synthetic peptides.
Calcitonins are 32-amino acid peptide hormones involved in the regulation of calcium metabolism. Calcitonin participates with parathyroid hormone in the regulation of bone metabolism and the homeostatic regulation of blood calcium levels according to mechanisms that are not completely understood. Normal bone metabolism comprises a balance of osteolytic resorption and osteoblastic formation of new bone to fill the resorption spaces. Calcitonin appears to oppose the osteolytic activity of parathyroid hormone, acting directly to inhibit bone resoration by altering osteoclastic and osteocytic activity. Caicitonin may also enhance new bone formation by stimulation of osteoblasts.
Calcitonin has a normalizing effect on serum calcium levels. In the normal individual, bone resorption is minimal and exogenous calcitonin has no hypocalcemic effect. However, in some pathological conditions, unopposed bone resorption causes a release of calcium and alkaline phosphatase into the circulation, and the appearance of urinary hydroxyproline, resulting from the breakdown of collagen-containing bone matrix. According to physiological mechanisms, elevated serum calcium levels promote the secretion of calcitonin to exert a hypocalcemic effect.
In addition to inhibiting the destruction of bone, accelerating the formation of new bone and controlling calcemia, calcitonin reduces calciuria and fixes calcium within the cell. Calcitonin also has important analgesic properties.
Exogenous calcitonin is therapeutically useful in disorders wherein bone turnover or resorption is accelerated. One important disease of this type is osteoporosis, particularly the postmenopausal type, marked by a progressive loss of bone mass. The efficacy of calcitonin treatment in osteoporosis is determined by increased total body calcium. Another disease of this type is Paget's disease (osteitis deformans), a disorder characterized by excessive resorption of bone accompanied by the imbalanced formation of new (pagetic) bone which lacks the characteristic architecture of normal bone. Effective calcitonin treatment reduces the elevated serum levels of alkaline phosphatase and urinary hydroxyproline seen in individuals with this diserase. Benefits of calcitonin therapy in Paget's disease are indicated by radiologic evidence of bone remodeling, correlated with a reduced number of osteoclasts seen in bone biopsies, consistent with a decrease in bone resorption.
Calcitonin therapy is useful in treating hypercalcemia, a condition that can be life-threatening if it persights. Hypercalcemia occurs with primary hyperthyroidism and in malignant diseases, principally carcinoma and malignant myeloma. The condition can occur when tumors are metastatic to bone as well as in carcinoma without metastasis.
Calcitonin also provides relief from the bone pain that accompanies bone resorption as in Paget's disease, osteoporosis, osteolysis of malignancy, and osteoporotic vertebral fractures. Calcitonin pain relief activity appears to be distinct from the effect exerted by the hormone on bone. When calcitonin therapy is employed, for example, in the treatment of Paget's disease, the analgesic effect precedes any change in the biochemical markers of bone disease, and pain relief persists even in patients who experience renewed episodes of bone lesions (Gennari, C. and D. Agnusdei, Current Therapeutic Research 44(5):712-722 (1988)).
Calcitonins are found in a variety of vertebrate species including mammals, birds and fish. The hormone is secreted by the C cells, which are localized in the thyroid gland of mammals, and in the ultimobranchial glands in the lower vertebrates. Human calcitonin (hCT) has the following amino acid sequence: ##STR1##
Calcitonin shows considerable divergence in amino acid sequence between lower vertebrates and higher vertebrates, with highly conserved residues clustered at the two ends of the calcitonin molecule believed to be important for biological activity. For example, a 1-7 disulfide bridge and a C-terminal proline amide are invariate among all species. Several other invariate amino acid residues occur near the N-and C-terminal ends. The middle portion of the molecule, positions 10 to 27, which is thought to control the potency and duration of the peptide, is by contrast quite variable in amino acid composition. Breimer, L. H., MacIntyre, I., and Zaidi, M., Biochem. J. 255:377-390 (1988) have reviewed the structures and biological properties of calcitonin peptides from various species and this information is hereby incorporated by reference.
Natural calcitonin peptides vary widely in their potency in humans, those of certain nonhuman species appearing to be more potent than human calcitonin. Calcitonins that are ultimobranchial in origin, such as salmon, eel, and avian, are more potent than the thyroidal human or porcine calcitonins. Salmon, eel, porcine and human calcitonins are currently used clinically in humans for the treatment of Paget's disease, osteoporosis, hypercalcemias, including hypercalcemia of malignancy, and for bone pain.
The correlation of potency with the structure of the calcitonin peptides is not well understood. Improved potency may be due to an amino acid sequence which permits a peptide conformation that is more favorably bound to the hormone receptor (Marx et al., Science 178:998-1001 (1972). A conformation that is more flexible, a feature provided by smaller, less bulky amino acids, has been determined to affect biological activity (Epand et al., Biochemistry 25:1964-1968(1988)). The identical biological potencies of eel and salmon calcitonin may accordingly be explained on the basis of similar primary structures and similar flexibility.
An alternative basis for the relatively greater potency of nonhuman calcitonins may be that the amino acid sequences of these calcitonins, characteristic of particular species, offers greater resistance to metabolic degradation in the human body than human calcitonin, and for this reason has a more persistent effect (Habener et al., Nature(London) 232:91-92 (1971)). For example, salmon calcitonin remains potent for about six hours after administration, while human calcitonin remains potent for about two hours.
In spite of their higher potency, however, non-human calcitonins, such as the ultimobranchial calcitonins, are not entirely satisfactory for human clinical use, primarily because the variable, poorly conserved middle portion of these calcitonins acts as an immunogen in vivo. The resulting antibody production can therefore limit their usefulness.
Further, after administration to man by subcutaneous injection, all the natural calcitonins have a relatively short half life because, in spite of species differences which act to retard proteolysis by plasma enzymes, they are subject to rapid renal and tissue clearance as well.
It would be useful to have calcitonin peptides which are more effective in clinical use either because of greater stability in vivo, and/or higher potency and longer duration of action than the native hormones. It would also be useful to have calcitonin peptides which are less immunogenic than the native hormones. Furthermore, analogues with increased lipophilicity and hydrophobicity could have altered pharmacokinetics and possess improved parenteral, nasal or oral bioavailability.
Accordingly, it is an object of the invention to provide synthetic calcitonin peptides which are more effective and less antigenic in the treatment of human disease than native calcitonins from human and other species. It is also an object of the invention to provide methods for the synthesis of these calcitonin analogues.